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In vitro apoptosis and expression of apoptosis-related molecules in lymphocytes from patients with systemic lupus erythematosus and other autoimmune diseases.

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ARTHRITIS & RHEUMATISM
Vol. 40, No. 2, February 1997, pp 306-317
0 1997, American College of Rheumatology
306
IN VITRO APOPTOSIS AND EXPRESSION OF
APOPTOSIS-RELATED MOLECULES IN LYMPHOCYTES FROM
PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS AND
OTHER AUTOIMMUNE DISEASES
HANNS-MARTIN LORENZ, MATHIAS GRUNKE, THOMAS HIERONYMUS, MARTIN HERRMANN,
ALMUTH KUHNEL, BERNHARD MANGER, and JOACHIM R. KALDEN
Objective. To analyze factors related to apoptosis
in systemic lupus erythematosus (SLE) peripheral
blood mononuclear cells (PBMC) and to compare the
findings in SLE PBMC with those in normal donor
PBMC or PBMC from patients with other autoimmune
diseases.
Methods. PBMC from normal healthy donors or
patients with SLE, mixed connective tissue disease
(MCTD), rheumatoid arthritis (RA), or various vasculitides were isolated. The percentage of apoptosis after
activation through different signaling pathways was
quantified using propidium iodide staining. Protein
expression of Fas/APO-1 or bcl-2, and messenger RNA
(mRNA) expression of bcl-2, bcl-x,, bax, buk, Fas/APO-1,
Fas ligand (Fas-L), c-myc, mad, or max were determined.
Results. We confirmed previous findings of increased numbers of apoptotic cells in SLE PBMC
compared with normal donor cells after in vitro incubation. After activation of PBMC with CD28 monoclonal
antibody plus phorbol myristate acetate (CD28 MAb/
PMA), staphylococcal enterotoxin B (SEB), or phytohemagglutinin (PHA), the percentage of apoptotic cells
was unchanged (SEB) or diminished (CD28 MAb/PMA,
PHA) in SLE cells, and the difference between normal
donor and SLE cells was less pronounced. On the
mRNA level, expression of apoptosis-related gene products did not differ between SLE cells and normal donor
Supported by Deutsche Forschungsgemeinschaft grants
Lo437/3-1 (to Dr. Lorenz) and Sonderforschungsbereich 263.
Hanns-Martin Lorenz, MD, Mathias Griinke, MD, Thomas
Hieronymus, PhD, Martin Herrmann, PhD, Almuth Kiihnel, Bernhard
Manger, MD, Joachim R. Kalden, MD: University of ErlangenNuremberg, Erlangen, Germany.
Address reprint requests to Hanns-Martin Lorenz, MD, Institute for Clinical Immunology and Rheumatology, University of
Erlangen-Nuremberg, Gliickstrasse 4a, 91054 Erlangen, Germany.
Submitted for publication January 29, 1996; accepted in
revised form August 28, 1996.
cells. Expression of Fas/APO-1 protein was increased in
freshly isolated SLE T lymphocytes compared with
normal donor T lymphocytes, whereas bcl-2 protein was
up-regulated after a 3-day culture period. Cellular activation further increased bcl-2 protein levels, eliminating
differences between normal donors and SLE patients. In
RA cells, the percentage of apoptosis was similar to that
in normal donor PBMC, whereas results using cells
from patients with other autoimmune diseases (MCTD,
Wegener’s granulomatosis, Takayasu arteritis, polyarteritis nodosa) were comparable with those found
using SLE PBMC. Addition of growth factors such as
interleukin-2 (IL-2), IL-4, or IL-15 to culture medium
decreased the percentage of in vitro apoptosis in both
normal donor and SLE cells.
Conclusion. Based on these data, we conclude
that accelerated in vitro apoptosis and increased F a d
APO-1 and bcl-2 protein expression in SLE are nonspecific for the disease, and might be explained at least in
part by the increased in viva activation levels of PBMC
from patients with SLE, MCTD, or autoimmune vasculitides combined with in vitro incubation under “noninflammatory” conditions and growth factor withdrawal.
The pathogenesis of most autoimmune diseases
is unclear. Recently, much attention has been devoted to
the role of apoptosis in the pathogenesis of these
diseases, especially of systemic lupus erythematosus
(SLE), based on findings in murine SLE models using
the MRLllpr mouse, the gld mouse, and the bcl-2 or
bcl-x, transgenic mouse. Cloning and characterization of
the murine FaslAPO-1 gene (1,2) was followed by the
finding that the genetic defect in MLRllpr mice is in the
Fas/APO-1 molecule (3,4). This is caused by insertion of
a retrotransposon in the second intron of the Fas/APO-1
gene, leading to aberrant splicing and prevention of
IN VITRO APOPTOSIS IN AUTOIMMUNE DISEASES
membranous expression ( 5 , 6 ) . Fas/APO-1 is an
apoptosis-promoting cell surface antigen (7-10). Thus,
in MLRllpr mice, Fas/APO-1 signals cannot mediate
apoptosis and cytotoxicity, and elimination of autoreactive cells is thereby prevented (3,ll). This leads to
clinical features resembling those of human SLE (1116). After introduction of a Fas/APO-1 transgene in
MLRllpr mice, most of the autoimmune features do not
develop (17).
Before the cloning of Fas/APO-1 or the Fas
ligand (Fas-L), Allen et a1 (18) had already suggested
that lpr and gld are mutations of genes encoding an
interacting pair of molecules. Thus, after cloning of the
gene for Fas-L (19-22), it was quickly recognized that
the genetic defect in gld mice is a point mutation in the
extracellular domain of the Fas-L (15,23-25). In addition, it was shown that this abnormal Fas-L cannot
induce apoptosis in Fas/APO-l-expressing cells (23).
Gld mice develop a disease that is phenotypically similar
to that found in Zpr mice, characterized by autoantibody
production, immune complex nephritis, and lymphadenopathy (12).
Other mouse strains with clinical features similar
to those of human SLE are mice transgenic for bcl-2
(26,27) or bcl-x, (28). Bcl-2 is an intracellular, apoptosisinhibitory protein (29) thought to block apoptosis due to
its function in an antioxidant pathway and an oxygenindependent pathway (30-32). Bcl-2 and the Fas/APO- 1
system seem to be, at least in part, functionally linked,
since it has been shown that bcl-2 signals are able to
inhibit Fas/APO-l-induced apoptosis (33). Bcl-2 transgenic mice show a polyclonal expansion of B cells with
high titers of IgG and IgA (26). At -1 year of age, the
mice develop high titers of antinuclear antibodies
against antigens, e.g., double-stranded DNA (dsDNA),
histones, or Sm/RNP, and usually die of immune complex glomerulonephritis (26). The phenotype of bcl-x,
transgenic mice is essentially indistinguishable from that
of a bcl-2 transgenic animal (28).
Several molecules known to influence the induction of programmed cell death have been studied in cells
from human SLE patients compared with normal
healthy donor cells. Fas/APO-1, an apoptosis-inducing
protein, was found to be expressed in higher quantities
on SLE lymphocytes (34). However, 2 different groups
of investigators found the apoptosis-inhibiting protein
bcl-2 expressed in higher intensity as well (35,36),
whereas another group did not detect any differences in
bcl-2 expression between SLE and normal donor cells
(37). A putative role of apoptosis in the pathogenesis of
human SLE was especially supported by a report by
307
Emlen et a1 (38) describing accelerated in vitro apoptosis of SLE lymphocytes in contrast to lymphocytes from
patients with rheumatoid arthritis (RA) or from normal
donors.
How could an increased rate of in vivo apoptosis
participate in the pathogenesis of SLE or other autoimmune diseases? The most important consequence of
apoptosis, as opposed to necrosis, is that the cellular
membranes are preserved, until finally, the apoptotic
cell body is rapidly removed and degraded via phagocytosis (39) without induction of an inflammatory response
(40). Thus, under physiologic conditions, cellular constituents are not released and therefore cannot activate
immunocompetent cells (40,41). However, in theory, an
increased rate of apoptosis could lead to an overflow of
the phagocytic system with apoptotic cell bodies. Thus,
intracellular constituents, such as apoptotic DNA fragments, would be presented to and recognized as nonself
antigens by immunocompetent cells, leading to the
formation of autoantibody against intracellular particles
such as dsDNA.
In support of this scenario, Casciola-Rosen et a1
(39) reported that after ultraviolet (UV) irradiationinduced in vitro apoptotic cell death of keratinocytes,
most of the known SLE autoantigens are clustered
within surface blebs of apoptotic cells. This generated
high concentrations of known autoantigens within discrete subcellular packages. In another study, the same
group found that after induction of apoptosis by Sindbis
virus infection in HeLa cells, viral antigens and autoantigens cocluster exclusively in small surface blebs of
apoptotic cells (42). These blebs form antigenic structures of mixed viral and self origin and could define a
novel immune context. Thus, an immune response originally directed against the virus could easily react with
apoptotic particles. This could lead to formation of
autoantibodies against intracellular constituents and initiation of autoimmune disease.
The aim of the present study was to investigate
whether activation of SLE peripheral blood mononuclear cells (PBMC) through various signaling pathways could differentially influence in vitro apoptosis
compared with normal donor PBMC. This is of special
interest because viral or bacterial infection in SLE
patients is frequently followed by increased disease
activity. In addition, we measured expression of bcl-2,
FasiAPO-1, and other apoptosis-related gene products,
i.e., bcl-x, (43), b m (44), mud (45,46), m m (47-49), and
c-myc (46,49-51) or the recently cloned buk (52-54) and
Fas-L (22). In order to investigate whether accelerated
in vitro apoptosis is specific to SLE, we tested PBMC
LORENZ ET AL
308
from patients with other autoimmune diseases, such as
mixed connective tissue disease (MCTD), Wegener's
granulomatosis, Takayasu arteritis, and polyarteritis nodosa, with regard to in vitro apoptosis or expression of
apoptosis-related gene products.
PATIENTS AND METHODS
Patients with SLE (n = 25), RA (n = ll), MCTD (n =
15), polyarteritis nodosa (n = 4), Takayasu arteritis (n = 2),
and Wegener's granulomatosis (n = 5 ) were recruited from
our outpatient clinic. All patients fulfilled applicable American
College of Rheumatology criteria for the diagnosis of the
respective diseases. In parallel with patient PBMC, normal
donor cells were isolated and used as controls in each
experiment.
PBMC were immediately separated by Ficoll-Hypaque
(BAG, Lich, Germany) density gradient centrifugation,
washed twice with phosphate buffered saline (PBS), and
resuspended to 4 X 10' cells/ml in RPMI 1640 containing 4
mM L-glutamine, 100 unitsiml penicillin G, 0.1 mgiml streptomycin (all from BioWitthaker, Verviers, Belgium), 15 mM
HEPES buffer (Roth Chemical, Karlsruhe, Germany), and
10% heat-inactivated (30 minutes, 56°C) fetal calf serum
(FCS; Gibco BRL, Eggenstein, Germany) or 10% autologous
serum. For generation of phytohemagglutinin (PHA) blasts,
PBMC from normal donors were stimulated with PHA (1
pgiml; Sigma, Deisenhofen, Germany) for 6 days, followed by
stimulation with interleukin-2 ([IL-21 10 unitsiml; Boehringer
Mannheim, Mannheim, Germany) for an additional 2 days.
Cells were washed extensively and incubated in RPMI 16401
10% FCS under the conditions indicated.
Reagents and monoclonal antibodies (MAb) were as
follows: CD28 MAb 9.3 (IgG2a, 15,000 dilution of ascites;
provided by Dr. J. Ledbetter, Bristol-Myers Squibb, Seattle,
WA), staphylococcal enterotoxin B (SEB; 10 ngiml), PHA (1
pgiml), phorbol myristate acetate (PMA; 1 ngiml) (all chemicals from Sigma), IL-2 (10 unitsiml; Boehringer Mannheim),
IL-4 (10 ng/ml; Genzyme, Boston, MA), and IL-15 (10 ngiml;
Pepro Tech EC, London, England).
Double staining for CD3 and FasiAPO-l was performed by incubating 4 X los PBMC in RPMI 1640110% FCS
with FASIAPO-1 MAb (IgG3; provided by Dr. P. Krammer,
Heidelberg, Germany) for 30 minutes at 4°C. After 3 washes in
cold PBS/O.S% bovine serum albumin (BSA; Sigma), cells were
incubated in 5 pl fluorescein isothiocyanate (F1TC)-labeled
anti-mouse Ig polyclonal antibody (Dianova-Immunotech,
Marseilles, France) for 30 minutes at 4°C. After 3 washes in
PBS/O.5% BSA, cells were resuspended in 5 pl of phycoerythrin (PE)-labeled CD3 MAb (Dianova-Immunotech) for
30 minutes at 4°C. After 3 additional washes, cells were fixed in
PBS/l% paraformaldehyde (Sigma, Diesenhofen, Germany),
and fluorescence was quantified using a Coulter fluorimeter
(Hamburg, Germany). For staining of intracellular bcl-2 protein, 1 X 10" cells were stained with PE-labeled CD3 MAb and
fixed in PBSi0.25% paraformaldehyde as described above. Cell
membranes were permeabilized by incubation of cells in 50%
methanol for 1 hour, and an FITC-labeled bcl-2 MAb (Dako
Diagnostica, Hamburg, Germany) was added. Cells were incu-
bated for 30 minutes at 4°C. After 3 washes in PBS, fluorescence was quantified with a Coulter fluorimeter.
The percentage of apoptotic cells was quantified immediately after isolation of PBMC, or after an incubation
period of 3 days, according to a method published elsewhere
(55). Briefly, PBMC were lysed in a hypotonic buffer containing 0.1% sodium citrate and 0.1% Triton X-100 along with 50
pgiml propidium iodide (PI) for DNA staining. Fluorescence
was determined using a Coulter fluorimeter. Applying this
method, nuclei from non-apoptotic cells show a narrow peak
with high fluorescence intensity, representing cells with a
normal chromatin content (Go or G, phase of the cell cycle),
and some smaller peaks with higher fluorescence intensity,
representing cells with hyperdiploid DNA contents (S or M
phase of the cell cycle). In contrast, nuclei of apoptotic cells are
stained in lower intensity by PI, and can be detected in a broad
peak with lower fluorescence intensity than the diploid cells
(55). Particles without PI staining (necrotic cell fragments)
were excluded by appropriate gate and marker positioning.
The percentage of apoptotic cells was calculated as follows:
3'% apoptotic cells
=
% of cells in subdiploid peak
x 100
% of cells in subdiploid, diploid, and hyperdiploid peaks
For quantification of DNA synthesis, 1 X lo5 PBMC
were incubated in a total volume of 150 pl RPMI 1640/10%
FCS in 96-well U-bottom plates (Nunc, Kamstrup, Denmark)
with the reagents as indicated, in triplicate for 3 days, with 0.5
pCi 'H-thymidine (Amersham, Braunschweig, Germany) included for the last 6 hours. Cells were harvested and scintillation was counted.
For semiquantification of messenger RNA (mRNA),
1 X 10" PBMC were either lysed immediately after separation
of cells or were stimulated overnight with the reagents as
indicated and then lysed in 4M guanidinium isothiocyanate
(GITC) lysis buffer, containing 4M GITC, sarkosyl, and
2-mercaptoethanol (all from Sigma). RNA was extracted
through a phenolichloroform gradient, precipitated, and
washed with isopropanol and ethanol. Messenger RNA was
reverse transcribed and amplified as described previously (56).
Gene-specific primers and polymerase chain reaction
(PCR) amplification conditions were as follows: p-actin: upstream 5'-ATGGATGATGATATCGCCGCG-3'
(3 minutes
at 60"C), downstream 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGGGCC-3'; bcl-2: upstream 5'-GGTGCCACCTGTGGTCCACCTG-3' (1 minute at 60"C, 2 minutes at
72"C), downstream 5'-CCTCACTTGTGGCTCAGATAGG3'; h a : upstream 5'-AGGTCTTTTTCCGAGTGG-3' (3 minutes at 60"C), downstream 5'-CACAAAGATGGTCAGGGT3'; bcl-x,: upstream 5'-AGAAGGGACTGAATCGGA-3'
(3 minutes at 60°C), downstream 5'-ATGTGGTGGAGCAGAGAA- 3'; buk upstream 5 '-ACGACATCAACCGACGCrAT-3'
(2 minutes at 60"C, 3 minutes at 72"C), downstream
5'-ACCATTGCCCAAGTTCAG-3';
FasiAPO-1: upstream
5'-TTATCGTCCAAAAGTGTTA-3' (3 minutes at 6o"C), downstream 5'-TTCTGTTCTGCTGTGTCITG-3'; Fas-L upstream
5'-AAAAAAGGAGCTGAGGAAAG-3' (1 minute at 6WC, 1
minute at 72"C), downstream 5'-GTGCCTGTAACAAAGAATCATA-3'; c-myc: upstream 5'-7TCTCTGAAAGGCTCTCCIT-3' (3 minutes at 60"C), downstream 5'-TCTGGTTC-
IN VITRO APOPTOSIS IN AUTOIMMUNE DISEASES
351
*
ND
SLE
T
SEB
medium
PHA
CD28/PMA
Figure 1. Increased percentage of in vitro apoptosis in systemic lupus
erythematosus (SLE) peripheral blood mononuclear cells (PBMC).
SLE and normal donor (ND) PBMC (n = 25 in each group) were
isolated in parallel as described in Patients and Methods. Cells were
incubated for 3 days either in medium alone or with the stimuli as
indicated, and the percentage of apoptosis was determined. Data
shown are the mean t SD. = P < 0.05, SLE cells versus normal
cells. SEB = staphylococcal enterotoxin B; PHA = phytohemagglutinin; CD28IPMA = CD28 monoclonal antibody plus phorbol myristate
acetate.
*
ACCATGTCTCCT-3'; mad: upstream 5'-ATGAACATCCAGATGCTG-3' (3 minutes at 60°C), downstream
S'-CAGATCACCTGTGAGATA-3';
m a : upstream 5'-GACAAACGGGCTCATCATAAT-3' (3 minutes at 6O"C), downstream Sr-GTAGAGGCTG7TGTCTGA-3'; IL-4: upstream
S'-ATGGGTCTCACCTCCCAACTGCT-3' (3 minutes at
6O"C), downstream 5'-CGAACACTTTGAATATTTCTCTCTCAT-3'.
PCR products were semiquantified as described (56).
Briefly, complementary DNA was amplified for 28 cycles and
35 cycles (p-actin for 21 cycles and 28 cycles), PCR products
were electrophoresed in a 1.5% agarose gel, and bands were
visualized under UV light. Band intensities were graded as
described previously (56).
Statistical analysis was performed using the Wilcoxon
nonparametric test.
RESULTS
We first investigated whether activation by the
addition of stimuli such as CD28 MAb plus PMA, the
superantigen SEB, or the lectin PHA would differentially induce apoptotic cell death in SLE PBMC versus
normal donor PBMC in vitro. As illustrated in Figure 1,
we found a significantly increased rate of apoptotic SLE
PBMC after in vitro culture in medium alone. This
contrasted with the findings in PBMC analyzed imme-
309
diately after purification, in which there were no significant differences in the percentage of apoptotic cells
between SLE and normal donor PBMC (data not
shown). The percentage of apoptotic cells did not correlate with disease activity, treatment regimen, duration
of disease, or age or sex of the patients (data not shown).
After stimulation with any of the reagents listed above,
the frequency of apoptotic SLE PBMC was either
unchanged (SEB) or diminished (PHA, CD28 MAb plus
PMA) compared with culture in medium alone.
In order to test whether cells were efficiently and
equally activated after the addition of stimuli, we performed proliferation assays in parallel with the
apoptosis-related experiments. The mean ? SEM
counts per minute in normal donor PBMC and SLE
PBMC, respectively, with the various stimuli, were as
follows: medium 648 ? 66 and 370 ? 72 (P < 0.05), SEB
39,389 ? 2,262 and 26,038 5 2,798 ( P < 0.05), PHA
51,809 ? 3,617 and 32,577 t 6,220 ( P < 0.05), CD28
MAb plus PMA 22,381 t 3,237 and 20,646 ? 3,717 (P
not significant). Interestingly, activation of normal donor PBMC with SEB led to an increased rate of apoptosis compared with incubation in medium alone (difference with borderline significance, P = 0.045). Notably,
however, the difference in the rate of in vitro apoptosis
between SLE and normal donor cells was less evident
after stimulation compared with incubation in medium
alone (Figure 1). Only after cellular activation with
CD28 MAb plus PMA did differences between SLE and
normal donor PBMC remain statistically significant
(Figure 1). Based on these results, the subsequent
experiments were focused on incubation conditions using medium or CD28 MAbiPMA stimulation.
We next analyzed the expression of apoptosisrelated gene products, either on the mRNA level or on
the protein level. For this purpose, we semiquantified
mRNA of different apoptosis-related genes using a
reverse transcription-PCR (RT-PCR) method as explained in Patients and Methods. As illustrated in Figure
2, after stimulation of PBMC with either medium or
CD28 MAb plus PMA, no major differences between
SLE and normal donor PBMC in the amount of mRNA
for the various gene products could be detected. The
increased intensity of max PCR product as shown in
Figure 2 was not consistent throughout the study. Table
1 summarizes the mean ? SEM grades for all apoptosisrelated gene products tested in 10 different experiments
with normal donor and SLE PBMC. Differences between normal donor and SLE PBMC were not statistically significant. In addition, the data show that cellular
activation did not usually lead to an increase of mRNA
LORENZ ET AL
310
same size, indicating that transcription of the apoptosisrelated genes tested does not lead to major mRNA
deletions or insertions in SLE PBMC.
Sorting experiments indicated that in vitro culture of isolated T lymphocytes (sheep erythrocyte rosetting, plastic adhesion, purity -90%) or non-T cells (i.e.,
cells that did not bind to erythrocytes) led to apoptosis in
both cell types. However, differences in the percentage
of apoptotic cells between normal donor and SLE
PBMC were present only in the T lymphocyte fraction.
This was seen in studies of PBMC from 4 patients and 4
normal donors, with basically identical results (mean -+
SEM T lymphocyte fraction 25.2 -+ 10.1% in SLE
patients, 14.0 -+ 8.2% in normal donors; non-T lymphocyte fraction 11.7 f 4.9% in SLE patients, 9.8 & 5.2% in
normal donors). We therefore quantified surface expression of Fas/APO-1 protein on freshly isolated CD3-t T
lymphocytes. As shown in Figure 3 for a representative
SLE patient, Fas/APO-1 protein expression was upregulated on SLE T lymphocytes compared with normal
donor T cells, as indicated by an increased percentage of
Fas/APO-1-positive cells and an up-regulated mean fluorescence channel (as a measure for antigen density on
cellular membranes). The mean ? SEM percentage of
Fas/APO-1-positive T lymphocytes was 73 -+ 17% in SLE
patients versus 45 ? 5% in normal donors in (n = 10 in
each group; P < 0.05). Similar results were consistently
found after a 3-day culture in medium (data not shown).
p-actin
fasIAP0-1
fas-L
bcl-2a
bcl-x,
bax
bak
c-myc
mad
Table 1. Expression of messenger RNA for apoptosis-related genes
in peripheral blood mononuclear cells (PBMC) from systemic lupus
erythematosus (SLE) patients and normal donors"
max
Normal donor PBMC
ND
SLE
Figure 2. Expression of messenger RNA of apoptosis-related genes in
SLE and normal donor PBMC. Cells from SLE patients and normal
donors were isolated in parallel as described in Patients and Methods.
Cells were incubated overnight either in medium alone or with
CD28IPMA as indicated. Messenger RNA was isolated, reverse transcribed, and amplified as described in Patients and Methods. Results
shown are representative of 10 different experiments. See Figure 1 for
definitions.
quantities in apoptosis-related gene products. As a positive control, RT-PCR grades for IL-4 were included,
and the results indicated that activation of cells augmented quantities of IL-4 mRNA (Table 1). Similar
results were found for SEB- or PHA-stimulated cells
(data not shown). In addition, at least with the primers
used, SLE and normal donor PCR products ran at the
SLE PBMC
Gene
Medium
CD28
MAbIPMA
Medium
p-actin
FasIAPO-1
Fas ligand
bd-2
bcl-x,
bax
bak
c-myc
mud
max
Interleukin-4
3.9 t 0.24
2.4 F 0.27
3.0 F 0.33
3.1 t 0.24
3.0 t 0.22
2.5 ? 0.55
2 . 2 t 0.15
2.0 F 0.39
4.0 2 0.94
3.0 2 0.50
0.6 2 0.3
4.1 t 0.30
2.9 t 0.30
3.3 t 0.28
3.5 2 0.24
3.4 t 0.28
2.8 2 0.65
2.5 i 0.20
2.2 t 0.38
4.3 t 0.72
3.0 F 0.50
1.9 t 0.3
3.9 t 0.26
2.5 t 0.36
3.3 t 0.56
2.9 i- 0.23
3.1 t 0.33
2.7 2 0.81
2.0 t 0.10
1.8 t 0.49
3.7 t 0.33
3.0 t 0.40
0.7 t 0.33
CD28
MAbIPMA
~
~
~~
4.1 t 0.27
2.7 2 0.34
3.7 2 0.40
3.3 i 0.30
3.3 2 0.33
2.5 t 0.56
2.2 +- 0.18
1.8 t 0.46
4.0 2 0.47
3.0 t 0.40
2.0 ? 0.4
* Cells from SLE patient? and normal donors were isolated in parallel
as described in Patients and Methods. Cells were incubated overnight
either in medium alone or with CD28 monoclonal antibody plus
phorbol myristate acetate (CD28 MAWPMA) as indicated. Messenger
RNA was isolated, reverse transcribed, and amplified as described in
Patients and Methods. Reverse transcription-polymerase chain reaction (RT-PCR) bands were graded as described previously (56).
Values shown are the mean F SEM RT-PCR grades from 10 different
experiments.
IN VITRO APOPTOSIS IN AUTOIMMUNE DISEASES
ND
311
SLE
UI
a
I
8
0
.I
1088
Fas - FlTC
Fas - FlTC
Figure 3. Fas/APO-1 expression in SLE and normal donor T lymphocytes. Cells from SLE patients and normal
donors were isolated in parallel as described in Patients and Methods. After isolation, cells were double stained with
phycoerythrin (PE)-labeled CD3 and fluorescein isothiocyanate (F1TC)-labeled Fas/APO-1 as described in Patients
and Methods. Results shown are representative of 10 different experiments. See Figure 1 for other definitions.
Next we stained SLE and normal donor T lymphocytes or non-T lymphocytes (CD3- cells) for expression of intracellular bcl-2 protein, as described in
Patients and Methods. Freshly isolated cells from SLE
T-cells
SLE
0
0
0
0
301
.
g i
25
non-T-cells
ND
SLE
medium
CD28/PMA
'
medium
'
CDPWPMA.
Figure 4. BcI-2 expression in T lymphocytes and non-T lymphocytes
of SLE patients versus normal donors. Cells from SLE patients and
normal donors were isolated in parallel as described in Patients and
Methods. Cells were incubated for 3 days either in medium alone (n =
20) or with CD28PMA (n = 18) as indicated. Intracellular expression
of bcl-2 protein was determined on CD3+ T lymphocytes or CD3non-T lymphocytes as described in Patients and Methods. Data shown
are the mean and SD fluorescence intensities (log scale) in 15 different
experiments. = P < 0.05, SLE cells versus normal cells. See Figure
1 for definitions.
*
Figure 5. Increased percentage of in vitro apoptosis in PBMC of
patients with vasculitides other than SLE, but not in patients with
rheumatoid arthritis (RA). Cells from normal donors (n = 23),
patients with RA (n = l l ) , patients with mixed connective tissue
disease (MCTD) (n = 15), patients with vasculitis (polyarteritis nodosa
[n = 41, Takayasu arteritis [n = 21, or Wegener's granulomatosis [n =
5]), or patients with SLE (n = 25) were isolated as described in Patients
and Methods and were incubated for 3 days either in medium alone or
with CD28 monoclonal antibody (mAb)/PMA as indicated. The percentage of apoptosis was determined as described in Patients and
Methods. In experiments in which the cells were incubated in medium,
the percentages of apoptosis in cells from patients with MCTD,
patients with vasculitis, and patients with SLE all differed significantly
from that in cells from normal donors ( P < 0.05); in experiments in
which the cells were incubated with CD28 mAblPMA, the percentage
of apoptosis in cells from patients with SLE differed significantly from
that in cells from normal donors (P < 0.05). Horizontal bars indicate
group means. See Figure 1 for other definitions.
LORENZ ET AL
312
8
8
0
.l
a
1
.1
1000
bcl-2-FITC
neg-FITC
MCTD
--1.-
1000
neg-FITC
.l
1090
bcl-2-FITC
Figure 6. Bcl-2 expression in T lymphocytes of a mixed connective tissue disease (MCTD) patient versus a
normal donor (ND). Cells from the patient and the normal donor were isolated in parallel as described in Patients
and Methods. Cells were incubated for 3 days in medium alone. Intracellular expression of bcl-2 protein was
determined as described in Patients and Methods. To indicate background staining, a nonspecific isotypematched antibody was used instead of bcl-2 and CD3 monoclonal antibody. Results shown are representative of
13 different experiments. See Figure 3 for other definitions.
patients and normal donors did not differ in their
expression of bcl-2 (results not shown). However, as seen
in Figure 4, the mean fluorescence intensity for bcl-2 was
significantly increased in SLE T lymphocytes versus
normal donor T lymphocytes after a 3-day culture in
medium. We did not detect differences in bcl-2 expression in non-T lymphocytes (Figure 4). In some SLE
patients and normal donors, we performed double stainings for bcl-2 and CD14 (monocytes) or CD19 (B
lymphocytes), but did not detect significant differences
in the expression of bcl-2 in these cell types (results not
shown). In addition, Figure 4 demonstrates that after
CD28 MAb/PMA stimulation, bcl-2 protein expression
was up-regulated in cells from both normal donors and
SLE patients. However, the difference in bcl-2 protein
expression between SLE and normal donor T lymphocytes was eliminated under these conditions. Identical
results were found for cells stimulated with SEB or PHA
(data not shown).
In order to test the specificity of these in vitro
results for SLE, we used RA PBMC as an additional
control. We did not find differences between normal
donor and RA PBMC in the rate of in vitro apoptosis
(Figure 5 ) . However, in contrast to more generalized
autoimmune diseases, R A represents a disease in which
most of the activated cells migrate into the joints as the
primary site of inflammation. Therefore, we also tested
PBMC of patients with MCTD. As shown in Figure 5 , we
found a significantly increased rate of apoptotic cells in
MCTD PBMC compared with normal donor PBMC
after 3 days of culture in medium. Under those test
conditions, the amount of apoptotic cells did not correlate with either the activity of MCTD or with therapy
(data not shown). Again, as in SLE PBMC (Figure l),
IN VITRO APOPTOSIS IN AUTOIMMUNE DISEASES
,
medium
CD28mAbIPMA
Figure 7. BcI-2 expression in T lymphocytes o f rheumatoid arthritis
(RA) or mixed connective tissue disease (MCTD) patients versus
normal donors. Cells from normal donors (n = 17) or patients with RA
(n = 4) or MCTD (n = 13) were isolated in parallel as describcd in
Patients and Methods. Cells were incubated for 3 days either in
medium alone or with CD28 monoclonal antibody (mAb)/PMA as
indicated. Intracellular expression of bcl-2 protein was determined as
described in Patients and Methods. Data shown are the mean and SD
fluorescence intensities (log scale). See Figure 1 for other definitions.
the difference in the percentage of apoptotic cells between MCTD and normal donor PBMC was decreased
after stimulation with CD28 MAb/PMA (Figure 5). We
next studied PBMC of patients with the primary vasculitides Wegener’s granulomatosis (n = 5 ) , polyarteritis
nodosa (n = 4), or Takayasu arteritis (n = 2) (collectively referred to as “vasculitis”). Again we found a
significantly increased percentage of apoptotic cells in
PBMC from these patients compared with normal donor
PBMC after 3 days culture in medium alone (Figure 5 ) .
As demonstrated for SLE and MCTD PBMC, this
difference was diminished after stimulation with reagents such as CD28 MAb plus PMA (Figure 5).
When we tested the expression of bcl-2 in MCTD
T lymphocytes, we found significant differences between
MCTD and normal donor T cells after 3-day culture in
medium (Figures 6 and 7), in parallel with the results
obtained with SLE T lymphocytes (Figure 4). Again,
after stimulation with reagents such as CD28 MAb plus
PMA, bcl-2 protein expression was up-regulated, eliminating the differences between normal donor and patient T lymphocytes (Figure 7). There were no differences in the expression of bcl-2 in MCTD non-T
lymphocytes (data not shown).
Because we demonstrated the occurrence of in
vitro apoptosis in PBMC of patients with all of the
autoimmune diseases studied except for RA, we hypoth-
313
esized that a growth factor deficiency might contribute
to the apoptotic death of activated T cells in vitro. We
therefore incubated SLE PBMC in IL-2-supplemented
medium. We found a markedly reduced rate of apoptotic cells as compared with the findings under culture
conditions in which medium alone was used (Figure 8).
The mean 5 SEM percentage of apoptosis in all experiments performed under these conditions (n = 10) was
as follows: normal donor PBMC, medium 13.7 f 2.1396,
IL-2 8.6 5 1.69%; SLE PBMC, medium 28.7 f 4.89%,
IL-2 14.8 -C 3.86% ( P < 0.05, normal donor PBMC
incubated in medium versus SLE PBMC incubated in
medium; P < 0.05, SLE PBMC incubated in medium
versus SLE PBMC incubated in IL-2). However, addition of IL-2 could not completely block induction of in
vitro apoptosis in normal donor and SLE PBMC, nor
could it eliminate the differences between normal donor
and SLE PBMC. After IL-2 together with IL-4 or IL-15
was added to SLE and normal donor PBMC, the reduction of apoptosis did not exceed that observed with IL-2
alone (data not shown). Thus, in order to test whether
lack of complete inhibition of apoptosis is characteristic
of preactivated cells, we added optimal concentrations
of IL-2 to normal donor PHA blasts. As in SLE PBMC,
growth factor supplementation could not completely
abrogate in vitro apoptosis in normal donor PHA blasts
(data not shown).
DISCUSSION
Based on recent findings as described above, our
work focused on the regulation of apoptosis in immunocompetent cells of patients with autoimmune diseases.
Emlen and colleagues (38) reported that an accelerated
and increased rate of in vitro apoptosis can be observed
in human SLE cells. These results were confirmed in the
present study, with the exception that the rate of in vitro
apoptosis did not correlate with clinical SLE activity in
our patients, whereas a relatively weak correlation ( P =
0.037, r = 0.365) was reported by E d e n et al. Interestingly, despite the different levels of responsiveness,
activation of cells with the reagents used did not increase
the incidence of apoptotic cell death in either SLE
PBMC or normal donor PBMC, with the notable exception of SEB activation in normal donor cells (borderline
significance, P = 0.045). In addition, cellular activation
clearly diminished differences in the rate of apoptosis
between normal donor and SLE PBMC.
When testing the expression of mRNA for several apoptosis-related genes, we did not find marked
differences in the expression of the apoptosis-related
LORENZ ET AL
314
medium
IL-2
ND
SLE
Figure 8. Inhibition of in vitro apoptosis after addition of interleukin-2 (IL-2). PBMC from SLE patients or
normal donors were isolated in parallel as described in Patients and Methods. Cells were incubated for 3 days
either in medium alone or with 10 unitsiml IL-2. The percentage of apoptosis was determined as described in
Patients and Methods. Results shown are representative of 10 different experiments. See Figure 1 for other
definitions.
gene products, as defined by the semiquantification
method used (56). However, to further substantiate
these findings, Northern blot analysis of the gene products would be required, since the RT-PCR method is
generally not sensitive enough to detect small changes in
mRNA quantities. Due to the small number of cells
available, we could not perform Northern blot analysis
in this study. In addition, mRNA for RT-PCR was
derived from PBMC, a cellular mixture of several different cell types. In a small number of SLE patients and
normal donors, we have recently performed RT-PCR of
apoptosis-related gene products, comparing CD45RO +
and CD45RO- T lymphocytes. To date we have not
found consistent differences in the expression of
apoptosis-related gene products. Interestingly, cellular
activation had no influence on quantities of mRNA for
the apoptosis-related genes tested (in contrast to proinflammatory gene products such as IL-4), indicating
that the functional activity of these gene products might
be regulated mainly on the protein level.
In contrast to our findings, Boise et a1 (57) have
shown that stimulation of purified T cells with CD28
MAb along with CD3 MAb can increase mRNA expression of bcl-x,. However, these results are based on
experiments that differed in several ways from those
reported herein. First, Boise and colleagues examined
mRNA expression in pure T cells. In addition, their
mRNA results were based on Northern blot analysis,
which is more sensitive than RT-PCR for detecting
minor differences in mRNA quantities. Furthermore, in
their stimulation assay, pure T cells were activated by
plastic-cross-linked CD3 MAb with soluble CD28 MAb.
In contrast, in our cellular system, T cells were activated
in the presence of monocytes and B cells (as cells
providing accessory signals) with soluble CD28 MAb
plus protein kinase C activator PMA. These differences
in the test systems might explain the differing results. In
fact, in some patients we did find an up-regulation of
bcl-x, mRNA in our experiments, as indicated by the
increased average RT-PCR grade (Table 1). This was
also evident with Fas/APO-1, Fas-L, bcl-2, buk, and mud
mRNA (Table 1).Importantly, however, there were no
differences between normal donor and SLE cells.
We next tested surface expression of Fas/APO-1
and intracellular expression of bcl-2. We focused on
these proteins because 2 of the SLE mouse models
mentioned above are based on genetic alterations in
these genes. Similar to the findings in previously reported studies (34,58) we found more T lymphocytes
expressing Fas/APO-1 in freshly isolated SLE cells compared with normal donor cells. Notably, convincing data
showing that signaling through Fas/APO-1 can induce
apoptosis in SLE cells have recently been published
(34,58). This indicates that Fas/APO-1 protein seems to
IN VITRO APOPTOSIS IN AUTOIMMUNE DISEASES
be functional in SLE patients, in contrast to MRLilpr
mice. However, for induction of apoptosis through Fas/
APO-1, preactivation of SLE cells was still necessary,
despite the increased expression of Fas/APO-1 in freshly
isolated SLE cells (34). All of these data indicate that
signaling through FasiAPO-1 seems to be normally
controlled and regulated in SLE cells. Additionally, case
reports on children with functional Fas/APO-1 deficiency showed that these patients do indeed exhibit
autoimmune phenomena such as autoimmune hemolysis
(59,60), but do not develop classic SLE.
Expression of apoptosis-inhibitory protein bcl-2
on freshly isolated PBMC has been a matter of controversy, with 2 groups of investigators showing increased
bcl-2 expression in T cells but not B cells (58,61) and
another group showing unaltered bcl-2 quantities in
unfractionated SLE PBMC (37). In the present study,
we did not detect differences in bcl-2 protein expression
immediately after isolation of the cells (data not shown).
However, we did find that after 3 days of incubation in
medium, bcl-2 density was significantly up-regulated in
SLE T lymphocytes, despite the increased rate of in vitro
apoptosis. Importantly, Fas/APO-1 and bcl-2 protein
expression was up-regulated in both normal donor and
SLE cells after activation of cells with various reagents,
eliminating the differences between the 2 groups. This
suggests that up-regulation of both apoptosis-related
proteins can be interpreted as indicating cellular activation, rather than dysregulated programmed cell death.
Whether there is an imbalance of apoptosis-inducing
proteins and signals through FasiAPO-1 (or through
other apoptosis-inducing proteins) and apoptosisinhibitory proteins such as bcl-2 or bcl-x (or others),
leading to disturbed regulation of apoptosis and possibly
to clinical symptoms such as those found in SLE, remains to be elucidated. The finding of FasiAPO-1
up-regulation without significantly increased bcl-2 expression in freshly isolated SLE PBMC/T lymphocytes
could reflect such an imbalance. Further studies are
needed to clarify this.
In this context, 2 questions were of interest: first,
whether these in vitro data are specific for SLE or can
also be seen in cells from patients with other autoimmune diseases, and second, whether the rate of in
vitro apoptosis can be reduced in an inflammatory
milieu. There were no major differences in the amount
of in vitro apoptosis in RA and normal donor PBMC,
confirming results reported by Emlen et a1 (38). However, in RA, most of the activated T cells migrate into
the inflamed joints and are therefore not present in the
peripheral vessels. In contrast, in PBMC from patients
315
with autoimmune diseases other than SLE or RA, i.e.,
MCTD, Wegener’s granulomatosis, polyarteritis nodosa,
and Takayasu arteritis, we found significantly increased
rates of in vitro apoptosis. Our results clearly show that
accelerated in vitro apoptosis, as well as up-regulation of
bcl-2 protein, represent features that are nonspecific for
SLE, but can also be seen in autoimmune diseases
characterized by a different pattern of autoantibodies, or
in activated normal donor cells.
Furthermore, we tested whether in vitro T lymphocyte apoptosis could be diminished by the addition of
growth factors such as IL-2, IL-4, or IL-15. It is well
documented that not only growth factor-dependent cell
lines such as CTLL-2, but also activated normal donor T
lymphocytes such as PHA blasts, undergo apoptosis
after growth factor withdrawal. In this proinflammatory
milieu we were clearly able to decrease in vitro apoptosis
in both normal donor PBMC and SLE PBMC. However,
the rate of apoptosis was still considerably higher in
patient cells compared with nonactivated normal donor
PBMC. Of note, after activation of normal donor cells,
we found similar levels of apoptosis despite optimal
concentrations of IL-2, IL-4, or IL-15 present in the
medium. We conclude that there seems to be a “point of
no return” in the cellular signaling pathway leading to
apoptosis, where even the presence of growth factors
cannot rescue the cell from programmed cell death.
From this perspective, it does not seem surprising that
the rate of in vitro apoptosis in SLE PBMC cannot be
completely reduced to the levels in nonactivated normal
donor PBMC.
Based on the results presented herein, we conclude that one of the most probable reasons for increased in vitro apoptosis of PBMC from patients with
SLE and such autoimmune diseases as MCTD, Wegener’s granulomatosis, and others is the in vivo preactivation of PBMC combined with an in vitro incubation
under “noninflammatory” conditions and growth factor
withdrawal. This could also explain why the rate of
apoptosis in autoimmune PBMC was diminished after
stimulation, at least with PHA or CD28 MAb/PMA,
since cellular activation induces production of growth
factors such as IL-2. Moreover, we conclude that the
increased rate of in vitro apoptosis and up-regulation of
Fas/APO-1 and bcl-2 expression are clearly not disease
specific and might, in contrast, reflect a physiologic
mechanism in the course of cellular activation to balance
between proliferation and apoptotic death. Of course,
these data do not exclude the possibility that an in vivo
dysregulation of apoptosis or apoptosis-related immune
mechanisms is important in the pathogenesis of human
LORENZ ET AL
316
SLE. However, further study of the regulation of apoptosis in autoimmune cells is required in order to
substantiate this hypothesis.
ACKNOWLEDGMENTS
We thank Drs. P. Krammer and J. Ledbetter for
generously providing antibodies, and Ms Bliss MarczinkeCroonquist for critical reading of the manuscript.
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